Only some of insulin's diverse effects on cell metabolism can be explained by this hormone's modulation of adenyl cyclase activity. Our initial studies of insulin's action on the phosphorylation and the de novo synthesis of fat cell proteins suggest new biochemical mechanisms to explain insulin's physiological actions. New techniques of discontinuous gradient slab acrylamide gel electrophoresis and autoradiography now permit study of the effects of insulin, epinephrine, cAMP, and other agents on fat cell proteins with increased sensitivity and specificity. We have found that in the absence of adenyl cyclase stimulation, insulin stimulated the incorporation of (32p)-orthophosphate into a high molecular weight protein or polypeptide (IPP; about 140,000 daltons). IPP phosphorylation occurred: 1) in both fat pads and isolated fat cells; 2) at physiological insulin concentrations (25 micro Units/ml); 3) within 15 min after insulin addition; and 4) in the absence of glucose; IPP phosphorylation was partially antagonized by epinephrine (10 to the minus 6th power M) and cAMP (10 to the minus 4th power M). Epinephrine and cAMP specifically increased the incorporation of (32p) into a protein (EPP) with a molecular weight (60-65,000 daltons) consistent with a subunit of fat cell lipase. EPP phosphorylation was specifically antagonized by either lithium ion or insulin. Both IPP and EPP turn over their (32p)-groups more rapidly than the total cellular phosphoprotein. This is the first non-cAMP mediated protein phosphorylation specifically induced by insulin. The present proposal will investigate: A) the protein kinase involved in IPP phosphorylation; B) the stimulation mechanism for the protein kinase (direct or indirect via "second messengers"); C) the possible donor phosphate molecule for IPP phosphorylation (ATP, GTP, PEP); and D) the reciprocal relationships between the effects of insulin and other hormones on IPP and those on EPP. An understanding of the relationship of insulin stimulated IPP to normal physiology will describe a unique intracellular circuit which may be critical in the mediation of some heretofore unexplained physiological effects of insulin, and may be involved in the etiology of human diseases, such as diabetes mellitus.